RU2165260C2 - Derivatives of arabinofuranosylcytosine and pharmaceutical compositions containing thereof - Google Patents

Abstract

FIELD: organic chemistry, pharmacy. SUBSTANCE: invention describes novel derivatives of arabinofuranosylcytosine of the formula (I)

(Ara-C) where R₁ and R₂ are taken independently from hydrogen atom and C₁₈- and C₂₀-saturated and monounsaturated acyl groups under condition that R₁ and R₂ both are not hydrogen atom; if R₁ means hydrogen atom then R₂ does not mean eicosenoyl (cis, trans) when R₁ means eicosenoyl (cis, trans), oleoyl, stearoyl, eicosanoyl; if R₁ means eladoyl then R₂ does not mean eicosenoyl (cis, trans), stearoyl, eicosanoyl; if R₁ means oleoyl then R₂ does not mean eicosenoyl (cis, trans), stearoyl; if R₁ means eicosanoyl then R₂ does not mean stearoyl; if R₁ means stearoyl then R₂ does not mean eicosanoyl, oleoyl. Invention describes the use of derivative of Ara-C of the formula (I) for preparing pharmaceutical preparation for treatment of patients with solid tumors and lymphomas. Invention describes also pharmaceutical composition for treatment of patients with solid tumors and lymphomas. EFFECT: new derivatives indicated above, valuable antitumor properties. 12 cl, 21 dwg, 3 ex

Description

 The invention relates to certain nucleoside derivatives, which have been found to have valuable properties for the treatment of tumors.

Ara-C иногда обозначают как цитозар.Nucleoside derivatives are esters of 1 - β-D-arabinofuranosylcytosine (Ara-C) of formula A:

Ara-C is sometimes referred to as cytosar.

 Ara-C has long been known as a chemotherapeutic agent for the treatment of acute myelogenous leukemia, but has limited effectiveness against solid tumors (Fre et al., Cancer Res. 29 (1969), 1325-1332; Davis et al. Oncology, 29 (1974), 190-200; Cullinan et al., Cancer Treat. Rep. 61 (1977), 1725-1726). However, even in the treatment of leukemia, Ara-C found only limited use due to its very short biological half-life and its high toxicity.

 In order to overcome these difficulties, a number of researchers have obtained and tested prodrug derivatives of Ara-C. For example, Hamamuca et al. Investigated 3'-acyl and 3 ', 5'-diacyl derivatives of Ara-C (J. Med. Chem. 19 (1976) N 5, 667-674). These researchers obtained and tested various Ara-C derivatives with saturated or unsaturated ester groups containing from 2 to 22 carbon atoms, and they found that many of these compounds showed higher activity against L1210 leukemia in mice than the original nucleoside itself.

The work of Hamamura et al. and other works on prodrug analogues of Ara-C are described in Hadfield et al. in Advances in Pharmacology and Chemotherapy, 20, 1984, pages 21-67. When discussing the 5 'Ara-C esters, these authors concluded (p. 27) that:
"although many of these agents appear to act as very effective forms of Ara-C depot in mice, a similar effect on humans has not been shown."

 Although research continued on Ara-C-based prodrugs, including 3 ′ and 5′-acyl derivatives (see, for example, Rubas et al. In Int. J. Cancer, 37, 1986, pages 149-154, which tested liposome ready-made formulations, among other derivatives, 5'-oleyl - Ara-C against leukemia L1210 and B16 melanoma), to date, no such drugs have become suitable for clinicians.

 The mechanism of action of Ara-C is based on its enzymatic recognition as 2'-deoxyriboside and subsequent phosphorylation to nucleoside triphosphate, which competes with normal CTP (cytosine-5'-triphosphate) for incorporation into DNA. The 2'-hydroxyl group causes steric hindrance to the rotation of the pyrimidine base around the nucleoside bond. Bases of polyarabinonucleotides cannot “grow” normally, as polydeoxynucleotide bases do. Ara-C inhibits DNA recovery and DNA synthesis both by slowing downstream elongation and the movement of newly replicated DNA via a matrix-linked replication apparatus. The mechanism of action of Ara-C leads to "unbalanced growth" in dividing cells. Ara-C acts in the S phase of the cell cycle. For continuous inhibition of DNA synthesis and ultimately cell death, Ara-C must be present in a sufficiently high concentration for at least one cell cycle.

 The main reason why Ara-C is not used in the treatment of solid tumors is again the rapid elimination of cancer cells and plasma from the active drug. Obviously, it is not possible to achieve significant intracellular drug levels in the tumor tissue, even if the tumor in question is sensitive to Ara-C in vitro. Unexpectedly prolonged half-lives and altered tissue distribution of the products of this invention will be of great importance for the therapeutic effects of these products.

 Found as shown in FIG. 7, 8 and 9, which, unlike Ara-C itself, as well as other mono and diesters, 3 'and 5'-O-esters of Ara-C and some saturated and unsaturated fatty acids unexpectedly show high activity against various tumors.

 It is believed that the commonly used test model (injection of leukemia cells into the abdominal cavity and intraperitoneal treatment) is more comparable to the in vitro model than to the actual clinical situation, and may lead to the inability to determine the particularly valuable properties of the selected Ara-C esters used in the present invention, as will be described below.

More specifically, the 3'- and 5'-O esters that are used in the present invention are esters derived from saturated and monounsaturated C ₁₈ or C ₂₀ fatty acids.

где R₁ и R₂ независимо выбирают из водорода и C₁₈ - и C₂₀-насыщенных и мононенасыщенных ацильных групп, при условии, что R₁ и R₂ не могут быть оба водородом.Thus, the esters used in the present invention can be represented by formula I:

where R ₁ and R _{2 are} independently selected from hydrogen and C ₁₈ and C ₂₀ unsaturated and monounsaturated acyl groups, provided that R ₁ and R ₂ cannot be both hydrogen.

 The double bond of monounsaturated acyl groups can be either in the cis or trans configuration, although the therapeutic effect may vary depending on which configuration is used.

The position of the double bond in monounsaturated acyl groups, apparently, also affects the activity. Currently, esters having an unsaturation in the ω-9 position are preferred. (In the ω-system of nomenclature, the position (ω) of the monounsaturated fatty acid double bond is calculated from the terminal methyl group, for example, eicosenoic acid (C ₂₀ : 1 ω -9) has 20 carbon atoms in the chain and the only double bond is formed between atoms 9 and 10, counting from the terminal methyl group of the chain). Thus, it is preferable to use Ara-C esters derived from oleic acid (C ₁₈ : 1, ω -9, cis), elaidic acid (C ₁₈ : 1, ω -9 trans) and eicosenoic acid (C ₂₀ : 1, ω -9, cis) and (C ₂₀ : 1, ω -9, trans), and stearic acid (C ₁₈ : O), and eicosanoic acid (C ₂₀ : O).

 In the treatment of various tumors in accordance with the present invention, both 3'-O and 5'-O monoesters and 3 ', 5'-diesters can be used, but 5'-O monoesters are generally preferred. It is believed that 3 ′, 5′-O-diesters will be useful when lipophilic properties are favorable, for example, when absorbed or absorbed in lipid tissues.

The compounds of formula (1), where R ₁ and R _{2 are} independently selected from the group consisting of hydrogen, elaidoyl, oleoyl, stearoyl, eicosenoyl (cis or trans) and eicosanoyl, provided that R ₁ and R ₂ cannot be both hydrogen, oleoyl or stearoyl. R ₁ cannot be hydrogen when R ₂ is oleoyl or stearoyl, and R ₂ cannot be hydrogen when R ₁ is elaidoyl, oleoyl or stearoyl, are new compounds not previously described in the prior art.

More specifically, the new compounds of formula (1) are defined in Table A at the end of the description, where R ₁ and R ₂ are indicated.

 The factor limiting the use of Ara-C is its destruction by cytidine deaminase and deoxycytidine monophosphate (dCMP) deaminase with the formation of inactive metabolites. It was unexpectedly discovered that the monoesters of this invention are poor substrates for these deactivating enzymes. This difference may mean that these ether derivatives are more suitable than Ara-C itself for systemic or local treatment of malignant tumors, especially malignant tumors in RES (reticuloendothelial system) and CNS (central nervous system).

 This is clearly demonstrated in the model of brain leukemic metastases shown in FIG. 10, 11 and 12, and especially with the more aggressive B-cell lymphoma shown in figure 11, where Ara-C itself is deprived of activity.

 In the clinical treatment of myelogenous leukemia, the rapid deactivation of Ara-C is compensated by continuous infusion of it for 5-7 days to establish a reasonably stable therapeutically active level of Ara-C in plasma. It was found that after intravenous administration to rats of equimolar amounts of radiolabeled Ara-C and Ara-C-5'-elaidyl ether, a successful change in metabolic rate and excretion profile is achieved. As can be seen from the data of table 1 and FIG. 20, the introduction of Ara-C-5'-elaidyl ether gives both a higher initial concentration in whole blood and plasma, and a slower conversion to Ara-U. The deamination of Ara-C to Ara-U of the esters of this invention — an example of the administration of elaidate is given here — is observed to be much slower and while plasma levels of both Ara-C and Ara-U are below the detection limit in the analysis in at 48 hours, when pure Ara-C is administered, these two compounds can still be quantified at 72 hours after administration of Ara-C-5'-elaidate. As can be seen from table 2, the total excreted amount of Ara-U (AUC, 0-72 h) is the same for both compounds administered. In clinical situations, these results are reflected in a wider time interval of the therapeutically active concentration of Ara-C in the blood. In the in vivo leukemia model shown in FIG. 13, Ara-C and 5'-elaidin ester are compared and an anti-cancer effect similar to that achieved with Ara-C is demonstrated by administering a 1/20 molar dose of ester.

 If the similar toxicity profile found in the Ara-C clinic is observed with ester derivatives, the improvement in therapeutic index should be of the same order of magnitude (x20) as the improvement in dose / effect ratio.

 The main value in the treatment of leukemia and other diseases limited by the reticuloendothelial system (RES) is, of course, the time interval of the active concentration of the drug in the plasma, but in addition, the localization of the active compound in RES tissue (defined as the liver, spleen, lymph, lungs, intestinal walls and free phagocytic cells present, for example, in bone marrow and whole blood). It was observed (FIG. 21 and Table 1) that with the intravenous administration of equimolar amounts of Ara-C and Ara-C-5'-elaidyl ether, the concentration of the active drug in the tissues of RES is significantly higher and a longer time interval remains after administration of the ether derivative. The nature of the distribution and metabolism was investigated in more detail and the results for the liver are shown in figure 19. Therapeutically significant level of Ara-C is maintained for at least 72 hours after administration of the ester derivative. This may provide an opportunity to treat initial liver cancer or liver metastases of colorectal cancer, breast cancer, melanoma, or other forms of cancer. The treatment can be carried out as monotherapy or as a palliative / help treatment in combination with surgery, radiation or other chemotherapy.

 Elevated concentrations of Ara-C in other tissues are also observed, and this, combined with a smaller volume of distribution, may open the way for the treatment of Ara-C esters of forms of cancer that are usually not associated with treatment with Ara-C.

 In addition, it was unexpectedly found that the esters of this invention (Figure 15) stimulate to a large extent the activation of NFkappaB, while Ara-C does not give any stimulation. Stimulation is a biological action not usually observed with therapeutic chemical agents, especially with conventional cytostatic agents. This suggests that the Ara-C esters of this invention have a stimulating effect on certain immune factors, which explains the unexpected improvement in anti-cancer effects. This is especially important in the treatment of neoplastic diseases involving immunocompetent cells, such as leukemia and lymphomatous.

 The development of resistant cancer cells is a serious problem in modern cancer chemotherapy. It was found (figures 7-9) that the Ara-C derivatives of this invention show the same effect against cisplatin resistant cells (NHIK 3025 / DDP) and MDR resistant cells (A549), as well as against the corresponding non-resistant cell lines . It can be assumed that this is due to the fact that these derivatives are not substrates for the cellular mechanisms of drug efflux from the cell, such as the “MDR gp 120 pump,” responsible for the phenomenon known as multi-drug resistance.

C ₁₈ - and C ₂₀ - mono - and diesters Ara-C can be used according to the present invention for the treatment of a number of neoplastic tumors. A particularly promising effect on breast tumors, such as gliomas, and metastasis from other tumors, such as sarcomas, carcinomas, as well as leukemia, has been found. Gliomas are currently treated with surgery, radiation therapy, and cytostatic agents such as N, N-bis- (2-chloroethyl) -N-nitrosourea (BCNU). However, the prognosis of such treatments is very poor.

 The beneficial effects of the Ara-C esters of the present invention are also found in metastatic tumors such as carcinoma, sarcomas, leukemia and melanomas.

 The scope of the invention and its essential and preferred features are defined in the attached claims.

Biological action
Micellar formulation
A 1 mg / ml micellar formulation is prepared by mixing 1: 1 (w / w) Ara-C ester (in DMSO) and lecithin (in ethanol) in sterile water.

Clonogenic Agarose Analysis ¹
A biopsy was taken from the patient and immediately placed on the growth medium. Tumor tissue was pulverized mechanically and living cells were selected, chemotherapeutic test substance, BCNU (in water) and Ara-C, and Ara-C esters (in micelles) were added and the cells were cultured in soft agarose. Twenty-four hours before the end of the cultivation (7 days) ³ H thymidine was added. The activity of the test substance was thus quantified as the number of pulses per minute in a scintillation counter.

¹ G. Unsgaard et al., Acta Neurochir (Wien) (1988) 91: 60-66.

Figure 1
Showing the results obtained with glioblastoma taken from a patient. The same response pattern was found in 8 other glioblastoma biopsies. The graph shows an in vivo comparison of Ara-C and its 3'-Elaidil ether and 5'-Elaidil ether. Results are reported as% of untreated control. The value of 50% (CD ₅₀ ) is chosen as promising for use in the treatment of this actual cancer line. Compared with elaidil esters, the concentration of Ara-C, higher than 10 ^1.5 , necessary to obtain CD ₅₀ , is not practical here.

Figure 2
Showing the results obtained with the same glioblastoma as in figure 1. The graph shows a comparison of radiation therapy and chemotherapy (BCNU). A radiation dose higher than 10 gray (Gy) required to obtain a CD ₅₀ is not practical in therapy. A comparison of figures 1 and 2 shows that the concentration of BCNU required to obtain CD ₅₀ , approximately 10 times higher than the concentration that is required when using ester Ara-C, but is acceptable comparable to the concentration of Ara-C itself.

Figure 3
Showing the results obtained with a biopsy taken from metastases of melanoma of the brain. The difference between Ara-C and 3'- and 5'-elaidil esters is not as pronounced as with glioma, but still 10 orders of magnitude higher.

Figure 4
BCNU activity in relation to the melanoma cell line is shown. Compared to Ara-C esters, a CDNU concentration of large 1x10 ² is required to produce CD ₅₀ .

Figure 5
This graph shows the results with metastatic carcinoma of the brain. These cancer cells are more resistant to chemotherapy, but the difference between Ara-C and Ara-C esters is still present.

Figure 6
The results of BCNU treatment of carcinoma (lung) metastases that are presented here are similar to those already demonstrated with other cell lines.

As for the other cell types studied, there appears to be a clear difference in the activity of the Ara-C, Ara-C, and BCNU esters. Activity 1 · 10 ^{2 is} very promising for therapy. The data obtained show that 5'-esters are more active than 3'-esters.

Cell Inactivation - Colony Ability
Cell inactivation, measured by the loss of the ability to form colonies, was determined for several compounds.

 The cells used were established cell lines of in situ origin for human cervical cancer, NHIK 3025, NHIK 3025 / DDP, a cis-DDP-resistant variant of the same cells or A549 cell (human lung carcinoma). These cells were exposed to the test compound for 4 to 24 hours. The test compounds were administered as a micellar solution. The number of colonies was counted approximately 12 days after incubation.

Figure 7
The graph shows an in vitro comparison of the tested compounds Ara-C, Ara-C-5'-elaidil ether, Ara-C-5'-stearyl ether, Ara-C-5'-eicosene ether and Ara-C-5'-petroselin ether . The results are shown as the dose needed to reduce cell survival by 90% with respect to the untreated control. As can be seen from the graph, there is a significantly higher inactivation of cells after exposure to these esters compared to Ara-C itself. The dose of the modifying factor at a survival level of 10% is in the range of 3 to 5 for Ara-C esters compared to Ara-C, which means that in order to obtain the same reduced ability to form colonies as observed for esters, a dose of Ara- is required C is 3-5 times higher.

Figure 8
The results of processing NHIK 3025 / DDP cells for 4 hours were obtained. An enhancement of action was observed in NHIK 3025 cells, similar to the detected enhancement of the action of Ara-C-5'-elaidate ester compared to the action of Ara-C. The enhanced effect is not dependent on cis-DDP resistance.

Figure 9
The graph shows the comparative in vitro colony ability of A549 cells (human lung carcinoma cells) for the tested compounds Ara-C, Ara-C-5'-Elaidyl ether, Ara-C-5'-stearyl ether, Ara-C- 5'-eicosene ester and Ara-C-5'-petroselin ester. Cells were exposed for 24 hours. The highest inactivation was observed for Ara-C-5'-stearyl ether, but an increased effect was also observed for elaidil and petroselin esters.

 Human Raji B lymphoma cells are a model of leptomeningitis carcinomatosis in nude rats.

The model used is a model of a tumor in nude rats for leptomeningitis tumor growth, 1 · 10 ⁶ cells of the Raji B-cell tumor line were injected into the cerebrospinal fluid through cisterna magna (cm) (the space between the arachnoid and the transverse slit of the cerebellum) of nude rats aged 4 -5 weeks. The animals developed neurological symptoms after 12-14 days if they were not treated. Anesthetized animals were treated with an intracerebral injection of 40 μl in cisterna magna 3 or 4 boluses. Processing began 1 day after cell inoculation. The tested compounds were Ara-C-5'-Elaidyl ether (in micelles) and Ara-C. Ara-C was administered both at the maximum tolerated dose (MTD) and at a dose equal to the equimolar dose of Ara-C-5'-elaidyl ether. Control animals (treated with NaCl or empty liposomes (micelles without Ara-C esters)) developed symptoms of central nervous system disorder after about 14 days.

Figure 10
Injections of 3 boluses with Ara-C elaidate on days 1, 2, and 4 increase the latent period without symptoms by 135% compared with Ara-C, with an average day of death delayed from day 13 to day 30.5, as seen in FIG. 10. One rat was alive for more than 70 days, it was considered as cured. When opening it on day 76 there were no visible tumors. This increase in the duration of disease-free existence is superior to the results obtained with other therapeutic alternative agents tested in comparable models for various types of human tumors.

Figure 11
This figure shows the survival curves for an additional experiment with naked rats inoculated with Raji cells in the brain and treated with doses of 4 boluses. A single bolus daily dose was administered on day 1, 2, 3, and 4 in cisterna magna. As in the previous experiment, no effect was observed for Ara-C, both at the maximum tolerated dose of Ara-C (MTD), and at a dose of an equimolar dose of Ara-C-elaidate. The results for the group receiving Ara-C-elaidate were even more surprising than the results in the previous experiment. 3 out of 5 rats were still alive and had no symptoms on day 70. They were considered as cases of recovery. This is the most promising result. 5 out of 6 control rats died by 13 laziness. The sixth control rat had no reverse cerebrospinal fluid injection into the syringe after injection of tumor cells and had no neurological symptoms after 70 days. In accordance with the usual method, this animal is not taken into account in the results.

Human Molt 4 Lymphoma Cells - Model of Naked Rat Leptomeningitis Carcinomatosis
The model used is a model of a tumor in nude rats for leptomeningitis tumor growth. 1 · 10 ⁶ cells of the Molt 4 T cell tumor line were injected into the cerebrospinal fluid through cisterna magna (cm) nude rats aged 4-5 weeks. The animals developed neurological symptoms after 20-22 days if they were not treated. Anesthetized animals were treated with an intracerebral injection of 40 μl in cisterna magna with 4 bolus injections. Processing began 1 day after cell inoculation. The tested compounds were Ara-C-5'-Elaidyl ether (in micelles) and Ara-C. Ara-C was administered both at the maximum tolerated dose (MTD) and at a dose equal to the equimolar dose of Ara-C-5'-elaidyl ether. Control animals (treated with NaCl) developed symptoms of a disorder in the central nervous system after approximately 20 days.

Figure 12
12 shows survival as a function of time for rats that were injected with Molt 4 lymphoma cells and treated 4 times in cisterna magna into the brain. In this initial experiment, the onset of death for animals receiving Ara-C-Elaidate was delayed compared to animals receiving Ara-C, or with control. The number of animals per group was as follows: control (7), Ara-C-elaidate (3), and Ara-C (5).

A leukemia model using human Raji B lymphoma cells
SCID mice were injected with intravenous 1 · 10 ⁶ human Raji B lymphoma cells. Mice were treated on days 7, 9, 11, 13 and 15 after injection of tumor cells with either 20 mg / kg / day of Ara-C-elaidate or 200 mg / kg / day of Ara-C, or they were a control. In animals, as a result of tumor growth, hind legs paralysis developed. The average day of death for animals treated with various treatments is shown in FIG. 13.

Figure 13
This figure shows the average survival of SCID mice injected intravenously with human Raji B-lymphoma cells and either treated intravenously with a single injection of Ara-C elaidate or Ara-C on each of days 7, 9, 11, 13 and 15, or being control. These doses were 20 mg / kg Ara-C-elaidate and 200 mg / kg Ara-C. On an equimolar basis, the dose of Ara-C-elaidate, reduced by 20 times compared with the dose of Ara-C, increased average survival compared to control and animals treated with Ara-C. The number of animals in each group was 7.

Figure 14
This figure shows the average survival of mice infected intravenously with human Raji B-lymphoma cells and either treated intravenously with a single injection on each day 7-11 of Ara-C-Elaidat or Ara-C, or being a control. The average survival time was significantly prolonged for Ara-C-elaidate, when the treatment is repeated daily instead of conducting it every other day.

Activation of cell transcription factor NFkappaB
We used human SW480 colon adenocarcinoma cells stably transfected with the promoter CMV / enhancer-containing β-galactosidase gene. Activation of the transcription factor NFkappaB leads to an increased content of the β-galactosidase enzyme in the cytoplasm. The amount of β-galactosidase was quantified using optical density at 570 nm as a parameter. SW380 cells were incubated 2-3 days before exposure to the test compound for 4 hours. The cells were washed and prepared and the absorbance was recorded for different compounds.

Figure 15
After exposure to Ara-C, no β-galactosidase activity was observed (determined by measurement), while after exposure to Ara-C-elaidate, a significant increase in β-galactosidase activity was observed, as an increase in optical density at 570 nm. This indicates that an unexpectedly high induction of the transcriptional activating protein NFkappaB is obtained with Ara-C-elaidate. NFkappaB is involved in the gene control of a number of immune factors, and this activation by Ara-C-elaidate may explain the increased anti-cancer effect observed for Ara-C-elaidate. Stimulation of some immune cells by Ara-C elaidate can be expected, which can be especially interesting in the treatment of leukemia and lymphomas.

Antitumor activity of Ara-C-elaidate compared to Ara-C against murine lymphoma TLX / 5
20-25 g CBA mice were inoculated subcutaneously in the groin with 1 × 10 ⁵ TLX / ⁵ tumor cells on day O. Ara-C-elaidate or Ara-C was intraperitoneally administered on days 3, 4, 5, 6 and 7. Doses were in the range of 6.25-50 mg / kg / day. Used 5 mice for each treatment group and 10 control mice with tumors. Activity was determined by increasing life expectancy (ILS) relative to control.

Figure 16
This figure shows the% increase in life expectancy of mice having a TLX / 5 lymphoma tumor (average 5 per group) after treatment with Ara-C-elaidate or Ara-C, intraperitoneal treatment for 5 days. Ara-C was only active at a dose of 25 mg / kg, while Ara-C-elaidate was active at 12.5 mg / kg and 25 mg / kg. The maximum increase in life expectancy was 47.2% compared with 32.7% for Ara-C.

Antitumor activity of Ara-C-elaidate compared to Ara-C in SCID mice inoculated intraperitoneally with hemangiosarcoma cells
SCID mice were inoculated intraperitoneally with PV / 2b / 35 hemangiosarcoma cells. Mice were treated 5 days a week with 25 mg / kg / day of Ara-C-elaidate prepared in micelles, Ara-C-elaidate dissolved in DMSO (dimethyl sulfoxide), or Ara-C dissolved in PBS (physiological saline with phosphate buffer) . Control mice received empty micelles, DMSO or PBS, respectively. The animals were not treated from Saturday to Monday. Survival was the end point of the study.

Figure 17
Survival of SCID mice inoculated intraperitoneally with PV / 2b / 35 hemangiosarcoma cells. Survival was significantly increased for animals treated with Ara-C-elaidate. Increased survival compared to control was observed for both Ara-C-elaidate prepared in micelles and for Ara-C-elaidate dissolved in DMSO.

Figure 18
The results presented here were obtained by studying the effect of 5'-Ara-C-elaidyl ether on the growth of glioblastoma tumor in nude mice. Tissue culture of the glioblastoma cell line U-118 (Uppsala) was injected subcutaneously in nude mice. A small portion (2x2 mm) of the growing tumor was transplanted to new mice. Subcutaneous tumors showed somewhat different growth rates in different animals, but at a size of 4-6 mm, a micellar solution of 10 mg / ml Ara-C ester was injected into the tumor. Depending on the actual tumor size, the animals received the same relative amount of test substance. Control mice received aqueous saline. Tumor growth was recorded as relative tumor volume (RTV). The control tumor developed according to the normal growth model typical of this type of cancer. Tumor growth was completely stopped in the treated animals. In addition, the animals did not show symptoms of toxic side effects, which in the case of Ara-C is bone marrow damage with the development of anemia or bleeding, nor were any symptoms of central nervous system disorder noted.

Comparative pharmacokinetics, distribution, metabolism and excretion of ¹⁴ C-Ara-C-elaidate and ¹⁴ C-Ara-C, administered intravenously to male rats
¹⁴ C-Ara-C-elaidate (in micelles) or ¹⁴ C-Ara-C was administered intravenously to male rats at equimolar doses, 5 mg / kg for ¹⁴ C-Ara-C-elaidate and 2.4 mg / kg for ¹⁴ C -Ara-C. The plasma concentration of total radioactivity and metabolites was determined at different time points (see Tables 1 and 2). The tissue concentration of total radioactivity was determined for various tissues at different time points up to 120 hours after injection. Liver tissue was extracted and the concentration of metabolites was determined up to 72 hours after injection. The distribution of Ara-C-elaidate in tissues varied significantly compared to the distribution of Ara-C. The maximum concentrations in most tissues were significantly higher and were observed at later time points after administration of ¹⁴ C-Ara-C-alaidate, especially in whole blood / plasma, spleen, liver and lungs. The maximum concentrations in the muscles, salivary glands, skin, and bladder were lower. It was found that the dose ratio in whole blood at 0.08 hours after administration of ¹⁴ C-Ara-C-elaidate was 64.7%, significantly higher than the proportion present in the pulmonary circulation at this time after administration of ¹⁴ C- Ara-C (7.76%). Excretion through the renal system was much slower for the elaidate than for the Ara-C itself. Removal from tissues was significantly slower for ¹⁴ C-Ara-C-elaidate compared to removal from tissues injected with ¹⁴ C-Ara-C.

Figure 19
The dependence of the concentration in the liver of Ara-C-elaidate (P-Ara-C-el) and metabolites Ara-C (P-Ara-C) and Ara-U (P-Ara-U) on the time after injection of ¹⁴ C-Ara -C-elaidate in figure 15, as well as the dependence of the concentration of Ara-C- (Ara-C) and metabolite Ara-U (Ara-U) on time after injection of ¹⁴ C-Ara-C itself. Injection of Ara-C-elaidate caused a significantly increased and prolonged effect on the liver of rats, both Ara-C-elaidate and Ara-C, without detecting Ara-U for up to 24 hours. This strongly contrasted with the concentration of Ara-C in the liver after administration Ara-C as such. The concentration of Ara-C in the liver decreased to an undetectable level after 4 hours in the presence of the metabolite Ara-U at all time points.

Figure 20
Dependence of plasma Ara-C elaidate and Ara-C and Ara-U metabolites in plasma after intravenous administration of ¹⁴ C-Ara-C elaidate as a function of time, as well as plasma levels of Ara-C and Ara-U metabolite after intravenous administration of ¹⁴ C -Ara-C from time to time. The administration of Ara-C-elaidate led to a prolongation of the time of the presence of Ara-C in plasma at a detectable level for up to 72 hours after administration compared to 24 hours after administration of Ara-C. Ara-C metabolism in Ara-U is less prolonged and begins later in animals that received Ara-C-elaidate.

Figure 21
The dependence of the concentration of total radioactivity in tissue on time after intravenous administration of either ¹⁴ C-Ara-C-elaidate (P) or ¹⁴ C-Ara-C was shown. The tissues shown in the graph were liver, spleen, lungs, bone, and bone marrow. The concentration of radioactivity after injection of ¹⁴ C-Ara-C-elaidate was higher at all time points up to 120 hours for all relevant tissues.

 Ara-C esters of the present invention can be formulated with conventional carriers and excipients for administration.

 As the most promising treatment regimen for gliomas and other solid brain tumors, local administration of active compounds at the site of the tumor to be affected is currently being considered. For this purpose, the active compounds may preferably be present in the form of a lecithin micellar formulation. For example, the preferred treatment for brain metastases may be to administer the Ara-C ester formulation to the cerebrospinal fluid or to the tumor area using a metering pump or similar device.

 The Ara-C esters of the present invention can also be administered systemically, either enterally or parenterally.

 For enteral administration, the active compounds of the present invention may be present in the form of, for example, soft or hard gelatin capsules, tablets, granules, grains or powders, dragees, syrups, suspensions or solutions.

 For parenteral administration, Ara-C ester formulations in the form of solutions, suspensions or emulsions for injection or infusion are suitable.

 The preparation may contain inert or pharmacodynamically active additives, also known to specialists in the field of finished formulations. For example, tablets or granules may contain a number of binding agents, filler materials, emulsifying agents, carrier substances or diluents. Liquid preparations may be present, for example, in the form of a sterile solution. Capsules may contain, in addition to the active ingredient, a filler material or a thickening agent. In addition, additives may also be present to improve the taste and smell of the preparation, as well as substances commonly used as preserving, stabilizing, moisture-retaining and emulsifying agents, salts for varying the osmotic pressure, buffers and other additives.

 The dose of the administered drug of the present invention will vary in accordance with the method of use and route of administration, as well as the needs of the patient. In general, the daily dose for systemic therapy for an adult average patient will be about 0.1-150 mg / kg body weight / day, preferably 1-50 mg / kg / day. For topical administration, the ointment, for example, may contain from 0.1 to 10 wt.% Of the pharmaceutical formulation, in particular 0.5-5 wt.%.

 If desired, a pharmaceutical preparation containing Ara-C esters may contain an antioxidant, for example, tocopherol, N-methyltocopheramine, butylated hydroxyanisole, ascorbic acid or butylated hydroxytoluene.

 Combination therapy, i.e. therapy in which the administration of the Ara-C ester of the present invention is carried out in combination with other therapies, for example surgery, radiation treatment and chemotherapy. For example, a preferred treatment for brain tumors comprising a combination of surgery and Ara-C ester treatment of the present invention with systemic or local administration.

где Nu-OH означает Ara-C; О представляет кислород в положении 3' и/или 5' части сахара Ara-C; Fa представляет ацильную группу насыщенной или мононенасыщенной C₁₈- или C₂₀- жирной кислоты, и X может быть Cl, Br или OR', где R' представляет Fa, COCH₃, COEt или COCF₃.Ara-C esters used in the present invention can generally be obtained in accordance with the following reaction equation:

where Nu-OH means Ara-C; O represents oxygen at position 3 'and / or 5' of the sugar Ara-C; Fa represents an acyl group of a saturated or monounsaturated C ₁₈ or C ₂₀ fatty acid, and X may be Cl, Br or OR ′, where R ′ is Fa, COCH ₃ , COEt or COCF ₃ .

Thus, the reaction proceeds through acylation of the nucleoside. It is carried out using suitable reactive derivatives of fatty acids, especially acid halides or acid anhydrides. When an acid halide such as an acid chloride is used, a tertiary amine catalyst such as triethylamine, N, N-dimethylaniline, pyridine or dimethylaminopyridine is added to the reaction mixture to bind the liberated hydrohalic acid. The reaction is preferably carried out in a non-reactive solvent such as N, N-dimethylformamide or a halogenated hydrocarbon such as dichloromethane. If desired, any of the above tertiary amines used as catalysts can be used as a solvent, ensuring that it is present in a suitable excess. The reaction is preferably carried out at a temperature between 5 and 25 ^° C. After 24 to 60 hours, the reaction is substantially complete. The progress of the reaction can be monitored using thin layer chromatography (TLC) and suitable solvent systems. When the reaction is completed as determined by TLC, the product is extracted with an organic solvent and purified by chromatography and / or recrystallization from a suitable solvent system. When more than one hydroxyl group as well as an amino group is present in Ara-C, a mixture of acylated compounds will be obtained. The desired individual mono- or di-O-esters can be separated, for example, by chromatography, crystallization, extraction under supercritical conditions, etc.

When it is desired to produce a diester compound of formula 1, where R ₁ and R ₂ are the same acyl group, it is preferable to use the above method using the corresponding acid chloride in excess.

In order to obtain a diester compound of formula 1 where R ₁ and R _{2 are} different, it is preferable to first obtain a 3 ′ or 5′-monoester and then the monoester to be reacted with a suitable acyl chloride.

 The receipt will be further shown in the following working examples.

Example 1
5'-O- (Elaidoyl) -1-β-D-arabinofuranosylcytosine ^1,2 .

To a suspension of I Ara-C-HCl (1.007 g, 3.6 · 10 ³ mol) in 15 ml of dimethylacetamide (DMA) is added a solution of elaidoyl chloride (1.26 g, 4.2 · 10 ^-3 mol) in 5 ml of DMA and the mixture is stirred at 30 ^° C. for 22 hours. The solvent was evaporated under high vacuum and the residue was taken up in hot ethyl acetate and filtered. The crude product is treated with 2 M aqueous NaHCO ₃ , filtered and purified on a silica gel column using methanol (5-30%) in chloroform as an eluent system. The homogeneous fractions were recrystallized to give 1.31 g (72%) of the title compound as a white solid (mp. 133-134 ^° C).

¹ H NMR (DMSO-d ₆ , 300 MHz) δ: 7.58 (1H, d, H-6), 7.18 (2H, broad d, NH ₂ ), 6.20 (1H, d, H-5 ), 5.77 (1H, d, H-1 '), 5.65 (2H, m, OH-2' and OH-3 '), 5.47 (2H, m, CH = CH), 4, 43 (1H, m, H-5 ' ₁ ), 4.30 (1H, m, H-5' ₂ ), 4.1-4.0 (3H, m, H-2 ', H-3' and H-4 '), 2.45 (2H, t, CH ₂ -COO), 2.05 (4H, m, CH ₂ -C =), 1.63 (2H, m, CH ₂ -C-COO), 1.35 (20H, m, CH ₂ ), 0.97 (3H, t, CH ₃ ).

¹³ C NMR (DMSO-d ₆ , 75 MHz) δ: 172.8 (COO), 165.59 (C4-N), 155.05 (C = O2), 142.86 (C-6), 130, 11 (CH = CH), 92.54 (C-5), 86.23 (C-1 '), 81.86 (C-4'), 76.83 (C-3 '), 74.35 ( C-2 '), 63.77 (C-5'), 33.46. 31.95, 31.30, 29.03, 28.97, 28.85, 28.73, 28.52, 28.43, 28.36, 24.48 and 22.12 (CH ₂ ), 13, 97 (CH ₃ ).

Example 2
3'-O- (Elaidoyl) -1-β-D-arabinofuranosylcytosine ^2,3 .

A mixture of 2-hydroxyisobutyric acid (1.15 g, 12 · 10 ^-3 mol) and elaidoyl chloride (3.10 g, 10 · 10 ^-3 mol) was stirred at 50 ^° C. for 1 h. Thionyl chloride (1.5 ml) was added. , 21 · 10 ^-3 mol) and stirring was continued for 2 hours. The reaction mixture was kept at 50 ^° C. and reduced pressure (40 mmHg) for 14 hours. The obtained 2-elaidoyloxy-2-methylpropanoyl chloride was used without any or further purification and suspended in 13 ml of anhydrous acetonitrile. Cytidine (0.608 g, 2.5 · 10 ⁻³ mol) was added and the reaction mixture was stirred at 60 ^° C. for 24 hours. The solvent was evaporated and the residue was treated with ether. The crude product was stirred in 40 ml of a 1: 1 mixture of pyridine-ethanol at 80 ^° C. for 20 hours, after which it was evaporated to dryness and the product was purified on a silica gel column. The homogeneous fractions were recrystallized to give 0.446 g (35%) of the title compound as a white solid (mp. 164-166 ^° C.).

¹ H NMR (DMSO-d ₆ , 300 MHz) δ: 7.71 (1H, d, H-6), 7.2 (2H, brd, NH ₂ ), 6.1 (1H, d, H -5), 5.88 (1H, d, h-1 '), 5.81 (1H, d, OH-2'), 5.45 (2H, m, CH = CH), 5.18 (1H , m, OH-5 '), 5.06 (1H, dd, H-3'), 4.18 (1H, m, H-2 '), 4.01 (1H, m, H-4'), 3 75 (2H, m, H-5 '), 2.47 (2H, t, CH ₂ -COO), 2.06 (4H, m, CH ₂ -C =), 1.65 (2H, m, CH ₂ -C-COO), 1.35 (20H, m, CH ₂ ), 0.97 (3H, t, CH ₃ ).

¹³ C NMR (DMSO-d ₆ , 75 MHz) δ: 172.15 (COO), 165.6 (C4-N), 154.95 (C = 0), 142.72 (C-6), 130, 11 and 130.08 (CH = CH), 92.59 (C-5), 86.24 (C-1 ^' ), 82.75 (C-4'), 78.72 (C-3 '), 72.29 (C-2 '), 61.15 (C-5'), 33.43, 31.97, 31.30, 29.03, 28.99, 28.85, 28.73, 28, 53, 28.41, 28.36, 24.40, 22.12 (CH ₂ ), 13.97 (CH ₃ ).

Example 3. 5'-O- (cis-11-eicosenoyl) -1-β-D-arabinofuranosylcytosine
To a suspension of Ara-C-HCl (0.87 g, 3.1 · 10 ^-3 mol) in 30 ml of N, N-dimethylformamide is added a solution of cis-11-eicosenoyl chloride (1.06 g, 3.22 · 10 ^-3 mol) in 30 ml of DMA and the reaction mixture was stirred at 25 ^° C. for 24 hours. The solvents were evaporated under high vacuum and the residue was dissolved in 60 ml of boiling ethanol, to which 20 ml of water and 20 ml of a saturated solution of NaHCO _{3 were} added. The crude product is filtered at room temperature and dissolved in 100 ml of boiling ethanol (60% in water). The crude product was recrystallized from ethyl acetate to give 1.1 g (66%) of the title compound as a white solid.

¹ H NMR (DMSO-d ₆ , 300 MHz) δ: 7.45 (1H, d, H-6), 7.1 (2H, broad d NH ₂ ), 6.08 (1H, d, H -1 '), 5.65 (1H, d, H-5), 5.55 (2H, m, OH-2' and OH-3 '), 5.32 (2H, m, CH = CH), 4.25 (1H, m, H-5 ' ₁ ), 4.15 (1H, m, H-5' ₂ ), 4.0-3.85 (3H, m, H-2 ', H-3 'and H-4'), 2.33 (2H, t, CH ₂ -COO), 1.95 (4H, m, CH ₂ -C =), 1,5 (2H, m, CH ₂ -C- COO), 1.25 (24H, m, CH ₂ ), 0.85 (3H, t, CH ₃ ).

¹³ C NMR (DMSO-d ₆ , 75 MHz) δ: 172.79 (COO), 165.59 (C4), 155.08 (C = 02), 142.78 (C = 6), 129.60 ( CH = CH), 92.52 (C-5), 86.21 (C-1 '), 81.82 (C-4'), 76.75 (C-3 '), 74.25 (C- 2 '), 63.76 (C-5'), 33.41, 31.30, 29.11, 28.85, 28.72, 28.60, 28.42, 26.57, 24.46, 22.11 (CH ₂ ), 13.94 (CH ₃ ).

Sources of information
1. DT Gish et al., J. Med. Chem. 14 (1971) 1159.

 2. E.K. Hamamura et al., J. Med. Chem. 19 (1976) 667.

 3. E.K. Hamamura et al., J. Med. Chem. 19 (1976) 654.

Claims (12)

1. The use of the derivative Ara-C of the formula I

where R ₁ and R _{2 are} independently selected from hydrogen and C ₁₈ and C ₂₀ unsaturated and monounsaturated acyl groups, provided that R ₁ and R ₂ are not both hydrogen;
when R ₁ is hydrogen, then R _{2 is} not elaidoyl, eicosenoyl (cis, trans);
when R ₁ is eicosenoyl (cis, trans), then R _{2 is} not hydrogen, elaidoyl, eicosenoyl (cis, trans), oleoyl, stearoyl, eicosanoyl;
when R ₁ is elaidoyl, then R _{2 is} not eicosenoyl (cis, trans), stearoyl, eicosanoyl;
when R ₁ represents oleoyl, then R _{2 is} not eicosenoyl (cis, trans), stearoyl;
when R ₁ is eicosanoyl, then R _{2 is} not stearoyl;
when R ₁ is stearoyl, then R _{2 is} not eicosanoyl, oleoyl,
as a treatment for solid tumors and lymphomas. 2. The use of the Ara-C derivative of formula I according to claim 1, wherein R ₁ and R _{2 are} independently selected from hydrogen and saturated or ω-9 monounsaturated C ₁₈ and C ₂₀ acyl groups. 3. The use of the Ara-C derivative of formula I according to claim 2, wherein R ₁ is hydrogen. 4. The use of the Ara-C derivative of formula I according to claim 3, wherein R ₂ is an elaidoyl or eicosenoyl (cis, trans) group.  5. The use of the Ara-C derivative of formula I according to any preceding paragraph as an agent for topical treatment of solid tumors or lymphomas.  6. The use of the Ara-C derivative of formula I according to any one of claims 1 to 4 as an agent for systemic treatment of solid tumors or lymphomas.  7. The use of the Ara-C derivative of formula I according to any preceding paragraph as an agent for treating solid tumors or lymphomas in the reticuloendothelial system (RES).  8. The use of the Ara-C derivative of formula I according to any one of claims 1 to 6 as an agent for treating solid tumors or lymphomas in the central nervous system (CNS).  9. The use of the Ara-C derivative of formula I according to any preceding paragraph as an agent for treating metastatic tumors. 10. Derived Ara-C of the formula I

where R ₁ and R _{2 are} independently selected from the group consisting of hydrogen, elaidoyl, oleoyl, stearoyl, eicosenoyl (cis or trans) and eicosanoyl, provided that R ₁ and R ₂ cannot be both hydrogen, oleoyl, elaidoyl, stearoyl or eicosanoyl; R ₁ cannot be hydrogen when R ₂ is oleoyl or stearoyl, and R ₂ cannot be hydrogen when R ₁ is elaidoyl, oleoyl, stearoyl or eicosanoyl.  11. The compounds of formula I of claim 10 to obtain a pharmaceutical composition for the treatment of solid tumors and lymphomas.  12. A pharmaceutical composition for treating solid tumors and lymphomas, comprising the Ara-C derivative of formula I of claim 10, and a pharmaceutically acceptable carrier or excipient.

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